In this Project, we will characterize synaptic, cellular and biochemical phenotypes of high-risk Alzheimer Disease (AD) mutations in human induced neuronal (iN) cells derived from iPS cells. We believe that the new advances in pluripotent stem cell biology and epigenetic reprogramming will provide an important breakthrough as they allow the genetic modification and functional evaluation of human neurons. Therefore, it is now possible to functionally interrogate risk mutations and study their cell biological effects in huma neurons. In particular, recent advances of gene targeting tools in human induced pluripotent stem (iPS) cells and our recent development of rapid methods that generate fully functional induced neuronal (iN) cells from iPS cells provide ideal conditions to begin to apply this technology to disease modeling for brain diseases such as AD. We will introduce into control iPS cell line derived from a well-characterized healthy normal subject conditional mutations that confer high risk for AD. Mutations will be introduced using homologous recombination in a protocol that we have developed in preliminary studies, and the conditionally mutant iPS cells will then be converted into precisely matched wild-type and mutant iN cells. Mutant and control cells will be characterized for Ab and Tau biochemistry and importantly for detailed synaptic properties. We believe the focus on the precise synaptic characterization represents a key innovative factor of our proposal as synaptic dysfunction may be much more sensitive than other cell biological assays such as cell death. Finally, we have confirmed in our iN cell/ astrocyte co-culture system that ApoE is primarily produced by the glia and that ApoE is a critical mediator of the glia-induced synaptic maturation of primary neurons and human iN cells, with possibly different effects of ApoE3 and ApoE4. Building on these results, we propose to evaluate in this specific aim the precise effects of ApoE3 and ApoE4 on synaptic maturation in wild type and APP-mutant iN cells generated in Aim 1, with the goal of gaining insight into the role of ApoE4 in AD pathology. Applied together, these specific aims will allow us to perform a well-controlled assessment of the effect of AD-associated APP mutations on the properties of human neurons and their synapses.